Assays for the serum level of Vitamin B12 have proven to be extremely challenging to develop. This is due primarily to the high sensitivity required, on the level of picograms, as well as the fact that normal serum contains endogenous B12 binders. These binders must be treated to release the B12 prior to the assay; such treatment is quite harsh and generally requires a separate step to accomplish it. Generally, the treatment involves the use of either heating to elevated temperatures (100.degree. C.), commonly termed "boil" assays, or the use of strong chemical agents, "no-boil" assays. One example of a no boil assay is presented in U.S. Pat. No. 4,300,907 to Mansbach et al.
Because of these requirements, until quite recently virtually all commercially available assays for B12 have been radio assays which utilize a radioactive isotopic labeled binding protein for detection. A number of other formats have been discussed in the literature including the use of chemiluminescent (Clin. Chem. Vol. 35, No. 6, p.1194, Abstract No. 609, (1989)), fluorescent (Clin. Chem. Vol. 37, No. 6 , p. 978, Abstract No. 326 (1991)), and color-labelled B12 (available from Ciba Corning) detectors. These assays utilize microbeads coated with B12 binding protein for detection, and, as such, are not compatible with many automated detection methods.
Additionally, enzyme linked assays utilizing alkaline phosphatase (Clin. Chem. Vo. 38, No. 6, p. 1095, Abstract 0691 (1992) and B galactosidase (Clin. Chem. Vo. 33, No. 6, p. 963, Abstract 403 (1989)) have been reported; however, both of these enzymes are quite large. Since the time required for competitive immunoassays is heavily dependent on the diffusion rate, and since the diffusion rate is approximately inversely proportional to the cube root of the molecular weight, the utility of these formats in such assays is limited.